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Electrochemical labelling of hydroxyindoles with chemoselectivity for site-specific protein bioconjugation

Nature Chemistry volume 16, pages 389–397 (2024)Cite this article

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Abstract

Electrochemistry has recently emerged as a powerful approach in small-molecule synthesis owing to its numerous attractive features, including precise control over the fundamental reaction parameters, mild reaction conditions and innate scalability. Even though these advantages also make it an attractive strategy for chemoselective modification of complex biomolecules such as proteins, such applications remain poorly developed. Here we report an electrochemically promoted coupling reaction between 5-hydroxytryptophan (5HTP) and simple aromatic amines—electrochemical labelling of hydroxyindoles with chemoselectivity (eCLIC)—that enables site-specific labelling of full-length proteins under mild conditions. Using genetic code expansion technology, the 5HTP residue can be incorporated into predefined sites of a recombinant protein expressed in either prokaryotic or eukaryotic hosts for subsequent eCLIC labelling. We used the eCLIC reaction to site-specifically label various recombinant proteins, including a full-length human antibody. Furthermore, we show that eCLIC is compatible with strain-promoted alkyne-azide and alkene-tetrazine click reactions, enabling site-specific modification of proteins at two different sites with distinct labels.

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Fig. 1: Electrochemical protein labelling strategies.
Fig. 2: Discovery and optimization of the eCLIC reaction.
Fig. 3: The substrate scope of eCLIC.
Fig. 4: Structural characterization of the eCLIC product.
Fig. 5: Functional conjugates of a full-length antibody using eCLIC.
Fig. 6: The eCLIC reaction is compatible with SPAAC and IEDDA.

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Data availability

Data supporting the findings of this study are available within the paper and its Supplementary Information. Crystallographic data for the structure reported in this Article have been deposited at the Cambridge Crystallographic Data Centre, under deposition no. CCDC 2179454 (19). Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/Search?Ccdcid=2179454. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Science Foundation (2128185 to A.C.) and by the NIH (R35GM134964 to E.W., R35GM136437 to A.C.). We thank M. Waegele (Boston College) for insightful discussions, B. Li (Director, XRD facility, B.C.) and T. Jayasundera (Director, NMR facilities, B.C.) for their assistance. The funding agencies had no role in study design.

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  1. These authors contributed equally: Conor Loynd, Soumya Jyoti Singha Roy.

Authors and Affiliations

  1. Department of Chemistry, Boston College, 2609 Beacon Street, Chestnut Hill, MA, USA

    Conor Loynd, Soumya Jyoti Singha Roy, Vincent J. Ovalle, Sarah E. Canarelli, Atanu Mondal, Delilah Jewel, Elise D. Ficaretta, Eranthie Weerapana & Abhishek Chatterjee

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Contributions

A.C., C.L. and S.J.S.R. designed the experiments and interpreted data. C.L. conducted bioconjugation experiments. S.J.S.R. generated the synthetic probes and performed structural characterizations. V.J.O. assisted with CV and earlier electrochemistry experiments. S.E.C. and E.W. performed trypsin digestion/MS and associated analysis. A.M. optimized the eCLIC modification of nanobody 5F7. D.J. assisted with flow cytometry analyses. E.D.F. assisted with bacterial expression of 5HTP-containing proteins. A.C., C.L. and S.J.S.R. prepared the manuscript. A.C. supervised the project.

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Correspondence to Abhishek Chatterjee.

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Competing interests

A patent application on the eCLIC technology reported here has been submitted. A.C. is a cofounder and senior advisor of BrickBio, Inc. The remaining authors declare no competing interests.

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Nature Chemistry thanks Sébastien Gouin, Wen-Bin Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary File 1

Crystal structure of NBoc–OMe–5HTP adduct with N,N-dimethylaniline

Source data

Source Data Fig. 5e

Raw data from MTT assay

Source Data Fig. 5c

Uncropped gels Fig 5c

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Loynd, C., Singha Roy, S.J., Ovalle, V.J. et al. Electrochemical labelling of hydroxyindoles with chemoselectivity for site-specific protein bioconjugation. Nat. Chem. 16, 389–397 (2024). https://doi.org/10.1038/s41557-023-01375-y

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